Investigation of derivatization reagents for the analysis of diarrhetic shellfish poisoning toxins by liquid chromatography with fluorescence detection
1994
Several derivatization reagents for the conversion of okadaic acid and related DSP toxins to fluorescent derivatives for analysis by liquid chromatography have been examined, viz. 9-anthryldiazomethane (ADAM), 1-pyrenyldiazomethane (PDAM), 4-diazomethyl-7-methoxycoumarin (DMMC), 4-bromomethyl-7-methoxycoumarin (BrMMC), 4-bromomethyl-7,8-benzcoumarin (BrMBC), 4-bromomethyl-7-acetoxycoumarin (BrMAC), and 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC). The ADAM reagent provides the greatest selectivity and sensitivity, but its application on a routine basis has been limited by its instability and cost. Improvement of this method was achieved through the production of ADAM in situ from the stable 9-anthraldehyde hydrazone. A detection limit of 30 ng/g hepatopancreas (equivalent to 6 ng/g whole tissue) was achieved. The other aryldiazomethane reagents were found to have insufficient reactivity. Of the bromomethylcoumarin reagents, BrDMC was found to have the greatest promise. The reagent is inexpensive and has excellent stability and purity. Quantitative derivatization may be achieved in a 2 hour reaction at 45 degrees C with N,N-diisopropylethylamine as a catalyst. Unfortunately, the lower reaction selectivity of BrDMC compared to that of ADAM limits its application to isolated toxins, plankton samples, and shellfish tissues with high levels of DSP toxins. The use of BrDMC for the determination of how toxin levels in shellfish tissues will require development of a more extensive clean-up prior to derivatization. Successful application of the ADAM and coumarin derivatization methods to real-world samples has been demonstrated.
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